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Behavioral and Brain Functions

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Association study of the vesicular monoamine transporter 1

(VMAT1) gene with schizophrenia in a Japanese population

Misty Richards1,2, Yoshimi Iijima1, Hitomi Kondo1, Tomoko Shizuno1,

Hiroaki Hori1, Kunimasa Arima3, Osamu Saitoh3 and Hiroshi Kunugi*1

Address: 1Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, 1878502, Japan, 2Albany Medical College, Albany, NY 12208, USA and 3Department of Psychiatry, Musashi Hospital, National Center of Neurology

and Psychiatry, Tokyo, 187-8502, Japan

Email: Misty Richards - RicharM@mail.amc.edu; Yoshimi Iijima - yiijima@ncnp.go.jp; Hitomi Kondo - kondomn@ncnp.go.jp;

Tomoko Shizuno - shizuno@ncnp.go.jp; Hiroaki Hori - hori@ncnp.go.jp; Kunimasa Arima - arimak@ncnp.go.jp;

Osamu Saitoh - osaitoh@ncnp.go.jp; Hiroshi Kunugi* - hkunugi@ncnp.go.jp

* Corresponding author

Published: 30 November 2006

Behavioral and Brain Functions 2006, 2:39

doi:10.1186/1744-9081-2-39

Received: 15 November 2006

Accepted: 30 November 2006

This article is available from:

? 2006 Richards et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (),

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Vesicular monoamine transporters (VMATs) mediate accumulation of

monoamines such as serotonin, dopamine, adrenaline, and noradrenaline from the cytoplasm

into storage organelles. The VMAT1 (alternatively solute carrier family 18: SLC18A1) regulates

such biogenic amines in neuroendocrine systems. The VMAT1 gene maps to chromosome

8p21.3, a locus with strong evidence of linkage with schizophrenia. A recent study reported that

a non-synonymous single nucleotide polymorphism (SNP) of the gene (Pro4Thr) was associated

with schizophrenia.

Methods: We attempted to replicate this finding in a Japanese sample of 354 schizophrenics and

365 controls. In addition, we examined 3 other non-synonymous SNPs (Thr98Ser, Thr136Ile,

and Val392Leu). Genotyping was performed by the TaqMan allelic discrimination assay.

Results: There was no significant difference in genotype or allele distribution of the three SNPs

of Pro4Thr, Thr136Ile, or Val392Leu between patients and controls. There was, however, a

significant difference in genotype and allele distributions for the Thr98Ser polymorphism

between the two groups (P = 0.01 for genotype and allele). When sexes were examined

separately, significant differences were observed in females (P = 0.006 for genotype, P = 0.003

for allele), but not in males. The Thr98 allele was more common in female patients than in female

controls (odds ratio 1.69, 95% CI 1.19�C2.40, P = 0.003). Haplotype-based analyses also provided

evidence for a significant association in females.

Conclusion: We failed to replicate the previously reported association of Pro4Thr of the

VMAT1 gene with schizophrenia. However, we obtained evidence for a possible role of the

Thr98Ser in giving susceptibility to schizophrenia in women.

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Behavioral and Brain Functions 2006, 2:39

Background

Vesicular monoamine transporters (VMATs) mediate

accumulation of monoamines such as serotonin,

dopamine, adrenaline, noradrenaline, and histamine

from the cytoplasm into storage organelles with an absolute dependence on a vacuolar ATPase-generated proton

gradient to transport the cationic amine substrates into

the storage organelle in exchange for protons [1-3]. There

are two isoforms of VMATs identified in rats and humans

[4-8]: VMAT1 (previously known as chromaffin granule

amine transporter; CGAT) and VMAT2 (alternatively designated as synaptic vesicle monoamine transporter;

SVMT). They are also the first and second members of the

solute carrier family 18 (SLC18A1 and SLC18A2, respectively). These proteins share 60% sequence identity; however, they demonstrate a range of differences in their

physiologic and pharmacologic properties. VMAT1 is

expressed primarily in neuroendocrine cells such as the

adrenal medulla and pineal gland, while VMAT2 is

expressed in all aminergic neurons in the mammalian

CNS [6,9,10]. The expression of the two isoforms in a

given cell type is usually, but not always, mutually exclusive [2,11]. Furthermore, the two isoforms differ in recognition of substrates (e.g., histamine) and sensitivity to

inhibitors such as tetrabenazine and methamphetamine

[12]. Since biogenic amines play critical roles in consciousness, mood, thought, motivation, cognition, perception, and autonomic responses, alterations in genes

encoding VMATs might play an important role in the

pathogenesis of neuropsychiatric diseases including schizophrenia.

With respect to the human VMAT2 gene, we previously

reported exon/intron boundaries, novel polymorphisms,

and association analysis with schizophrenia; however, we

did not find any polymorphism that resulted in an amino

acid change [13]. In addition, we failed to obtain evidence

for a significant association of the detected polymorphisms with schizophrenia [13]. The other VMAT,

VMAT1, is also an attractive candidate gene for schizophrenia not only because it plays a critical role in the

maintenance of monoaminergic endocrine systems but

also it maps to chromosome 8p21.3 [14], a locus with

strong evidence for linkage with schizophrenia [15-21]. In

accordance with the possible role of the VMAT1 gene in

schizophrenia, a recent study reported that an SNP in

exon 3 of the gene that results in an amino acid change

(277C > A resulting in Pro4Thr) was significantly associated with schizophrenia [22]. The C/C genotype

(homozygosity for proline residue at codon 4) occurred in

21.4% of the schizophrenic group and only 2.6% of the

control group. The A/A genotype (homozygosity for threonine), on the other hand, occurred in 28.6% of the schizophrenic group and 73.6% of the control group. Such a

dramatic difference in one polymorphism of the VMAT1



gene in a Caucasian population prompted us to attempt

replication of this finding in a Japanese population. In

addition, we examined other non-synonymous polymorphisms in the VMAT1 gene for association with schizophrenia.

Methods

Subjects

Subjects were 354 patients with schizophrenia (212

males, mean age of 44.0 years [SD 13.7]) and 365 healthy

controls (113 males, mean age of 39.7 years [SD 14.1]).

All subjects were biologically unrelated Japanese and

recruited from the same geographical area (Western part

of Tokyo Metropolitan). Consensus diagnosis by at least

two psychiatrists was made for each patient according to

the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria [23] on the basis of

unstructured interviews and information from medical

records. The majority of the patients (318 patients, 90%)

had a history of admission to a psychiatric hospital. Mean

age of onset was 24.4 years [SD 8.6]. Twenty-nine percent

of the patients (102 patients) had a family history of

schizophrenia spectrum disorders within the second

degree relatives. The controls were healthy volunteers

recruited from hospital staffs and their associates. Control

individuals were interviewed and those who had current

or past history of psychiatric treatment were not enrolled

in the study. The study protocol was approved by the ethics committee at the National Center of Neurology and

Psychiatry, Japan. After description of the study, written

informed consent was obtained from every subject.

Genotyping

Since genetic variations that result in an amino acid

change are most likely to alter function, we searched for

non-synonymous polymorphisms of the VMAT1 gene in

silico based on the NCBI dbSNP database and found 4

well-validated SNPs with a heterozygosity value of > 0.10.

They were rs2270641 (SNP1, 277C > A, Pro4Thr),

rs2270637 (SNP2, 560C > G, Thr98Ser), rs1390938

(SNP3, 674C > T, Thr136Ile), and rs17092104 (SNP4,

1441G > C, Val392Leu). The numbers of base and amino

acid positions were according to NM_003053 and

NP_003044, respectively. Venous blood was drawn from

the subjects and genomic DNA was extracted from whole

blood according to the standard procedures. The SNPs

were genotyped using the TaqMan 5'-exonuclease allelic

discrimination assay; the assay ID (Applied Biosystems)

for each SNP was C_22271506_10 for SNP1,

C_2716008_1 for SNP2, C_8804621_1 for SNP3, and

C_2715953_10 for SNP4. Thermal cycling conditions for

polymerase chain reaction (PCR) were 1 cycle at 95��C for

10 minutes followed by 50 cycles of 92��C for 15 seconds

and 60��C for 1 minute. Genotype data were read blind to

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Behavioral and Brain Functions 2006, 2:39

the case-control status. Ambiguous genotype data were

not included in the analysis.

Statistical analysis

Deviations of genotype distributions from the HardyWeinberg equilibrium were assessed with the ��2 test for

goodness of fit. Genotype and allele distributions were

compared between patients and controls by using the ��2

test for independence. These tests were performed with

the SPSS software ver 11 (SPSS Japan, Tokyo, Japan). Haplotype-based association analyses were examined with the

COCAPHASE software ver 2.4 [24]. The expectation-maximization (EM) and "droprare" options were used. Haplotypes with frequencies less than 3 % were considered to be

rare. We examined associations by permutation procedure

(10,000 replications) to determine the empirical significance.



Discussion

We failed to replicate the finding of Bly [22] who reported

a significant association between the Pro4Thr polymorphism (SNP1) of the VMAT1 gene and schizophrenia.

This discrepancy may be attributable to ethnic differences

in the effects of SNP1 between Caucasians and Asians. The

possibility of a type-II error is unlikely because our sample

size had a power of approximately 100% to detect the difference in the frequency of C/C genotype reported in Bly's

study (21.4% in patients and 2.6% in controls). Moreover, both the genotype and allele distributions of SNP1

were almost the same in our patients and controls. An

alternative possibility might be that the finding of Bly [22]

had arisen by chance due to the small sample size (28

schizophrenics and 38 controls) and thus obtained evidence of statistical significance was not strong (P = 0.036)

in spite of the marked difference in the frequency of C/C

genotype between patients and controls in his sample.

Results

Genotype and allele distributions of the examined SNPs

in patients and controls are shown in Table 1. The genotype distributions did not significantly deviate from the

Hardy-Weinberg equilibrium in patients and controls for

any SNPs. With respect to SNP1, there was no significant

difference in genotype or allele distributions between

patients and controls. Both genotype and allele distributions were approximately the same in the two groups;

therefore, we failed to replicate the finding of Bly [22]. For

the remaining SNPs, however, we found a significant difference in genotype and allele distributions of SNP2, but

not SNP3 or SNP4, between patients and controls. For

SNP2, the Thr98 (560C) allele was significantly more

common in patients than in controls (P = 0.01, odds ratio

= 1.39, 95% CI 1.09�C1.77). When men and women were

examined separately, genotype and allele distributions of

SNP2 significantly differ in females, but not in males,

between the two groups (Table 2). The excess of the Thr98

allele in patients was highly significant in females (��2 =

8.54, df = 1, P = 0.003, odds ratio = 1.69, 95% CI 1.19�C

2.40), whereas genotype and allele distributions were

quite similar in male patients and controls.

Pair-wise linkage disequilibrium values between neighbouring SNPs are shown in Table 3. Fairly tight linkage

disequilibrium was observed in any pair of the SNPs. We

obtained no significant difference in haplotype frequencies for two-, three-, or four-marker analyses between

patients and controls in males (data not shown). In

females, however, we obtained significant differences in

estimated haplotype distributions for any comparisons

when SNP2 was included in the analysis (Table 4). The

most significant result was obtained by the two-marker

haplotype (C-C) consisting of SNP2 and SNP3 (permutation P = 0.007).

When additional SNPs were genotyped, we found that the

98Thr (560C) allele of SNP2 was significantly increased in

schizophrenics compared to controls, although no significant results were obtained for SNP3 or SNP4. This significant excess of the 98Thr allele in patients was observed in

females, but not in males, suggesting that the Thr98 allele

has a sexually dimorphic effect of giving susceptibility to

schizophrenia. Considering that the frequency of the

98Thr allele was greater than the 98Ser allele, it might be

more appropriate to infer that the 98Ser allele has a protective effect against the development of schizophrenia.

Haplotype-based analyses also yielded several significant

differences in haplotype frequencies between female

patients and controls only when SNP2 was included in

the analysis, providing further support for the possible

role of SNP2 in female schizophrenia. However, since we

examined only non-synonymous SNPs that had been

deposited in the public database (dbSNP) and did not

perform polymorphism screening, we may have missed

unknown functional polymorphisms. It is possible that

such unknown polymorphisms nearby which are in linkage disequilibrium to the SNP2 might be "truly" responsible in giving susceptibility to schizophrenia.

The Thr98Ser polymorphism may affect the processing

and overall function of VMAT1 through altering cell signaling and protein trafficking pathways. The human

VMAT1 gene is composed of 18 exons which encode 525

amino acids [5]. There are 12 predicted transmembrane

domains in the VMAT1 secondary structure and a large

luminal loop between transmembrane domains 1 and 2.

The Thr98Ser polymorphism is located on this luminal

loop, in which there are three potential sites for N-linked

glycosylation (asparagines residues at codons 58, 87 and

104) [6]. This loop is the main site of N-glycosylation on

the VMAT1 protein, which is believed to regulate targeting

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Inter-SNP distance (bp)

Group

N

Genotype frequency (GF)

Allele frequency (AF)

Odds ratio (95% CI)

HWE

(df = 1)

7883394

SNP1 rs2270641

Exon 2

____

Pro4Thr

Exon 3

1639

Thr98Ser

Exon 3

114

Thr136Ile

Exon 13

31159

Val392Leu

C

A

45 (0.13)

153 (0.44)

153 (0.44)

243 (0.35)

459 (0.65)

0.83 �C 1.29

��2 = 0.48, P = 0.49

P = 0.95

P = 0.75

Controls

360

49 (0.14)

157 (0.44)

154 (0.43)

255 (0.35)

465 (0.65)

1.04

��2 = 0.78, P = 0.37

��2 = 0.11

��2 = 0.10

C/C

G/C

G/G

C

G

Patients

352

11 (0.03)

130 (0.37)

211 (0.60)

152 (0.22)

552 (0.78)

1.09 �C 1.77

��2 = 2.9, P = 0.09

P = 0.01

P = 0.01

Controls

362

28 (0.08)

144 (0.40)

190 (0.52)

200 (0.28)

524 (0.72)

1.39

��2 = 0.0, P = 0.92

��2 = 9.09

��2 = 7.00

C/C

T/C

T/T

C

T

Patients

352

188 (0.53)

135 (0.38)

29 (0.08)

511 (0.73)

193 (0.27)

0.70 �C 1.13

��2 = 0.46, P = 0.50

P = 0.44

P = 0.33

Controls

360

200 (0.56)

139 (0.39)

21 (0.06)

539 (0.75)

181 (0.25)

0.89

��2 = 0.24, P = 0.62

��2 = 1.62

��2 = 0.95

G/G

G/C

C/C

G

C

7850482

SNP4 rs17092104

A/A

351

7881641

SNP3 rs1390938

A/C

AF

(df = 1)

Patients

7881755

SNP2 rs2270637

C/C

Chi-square test

GF

(df = 2)

Patients

352

0 (0.00)

23 (0.07)

329 (0.93)

23 (0.03)

681 (0.97)

0.38 �C 1.34

��2 = 0.40, P = 0.53

P = 0.28

P = 0.29

Controls

363

0 (0.00)

17 (0.05)

346 (0.95)

17 (0.02)

709 (0.98)

0.71

��2 = 0.21, P = 0.65

��2 = 1.16

��2 = 1.13

Behavioral and Brain Functions 2006, 2:39

aChromosome position was according to the dbSNP database.

HWE: Hardy-Weinberg equilibrium

P values of < 0.05 are underlined.

Table 2: Genotype and allele distributions of SNP2 (Thr98Ser) in patients with schizophrenia and controls for each sex

N

Genotype distribution (frequency)

Allele distribution (frequency)

Patients

Controls

352

362

11

28

CC

(0.03)

(0.08)

130

144

GC

(0.37)

(0.40)

211

190

GG

(0.60)

(0.52)

��2

9.09

P

0.011

Patients

Controls

211

112

9

9

(0.04)

(0.08)

79

37

(0.37)

(0.33)

123

66

(0.58)

(0.59)

2.27

Patients

Controls

141

250

2

19

(0.01)

(0.08)

51

107

(0.36)

(0.43)

88

124

(0.62)

(0.50)

10.12

Total

C

HWE

��2

7.00

P

0.008

��2

2.90

0.01

P

0.089

0.921

(0.77)

(0.75)

0.20

0.655

0.70

1.31

0.404

0.252

(0.80)

(0.71)

8.54

0.003

3.26

0.39

0.071

0.534

G

152

200

(0.22)

(0.28)

552

524

(0.78)

(0.72)

0.322

97

55

(0.23)

(0.25)

325

169

0.006

55

145

(0.20)

(0.29)

227

355

Male

Female

HWE: Hardy-Weinberg equilibrium

Significant P values are underlined.

Page 4 of 6

Positiona

dbSNP ID

(page number not for citation purposes)



Table 1: Genotype and allelic distributions of the VMAT1 SNPs in patients with schizophrenia and controls

Behavioral and Brain Functions 2006, 2:39



Table 3: Pair-wise linkage disequilibrium between neighbouring SNPs in the VMAT1 gene

SNP1

rs2270641

SNP1

SNP2

SNP3

SNP4

SNP2

rs2270637

SNP3

rs1390938

SNP4

rs17092104

0.70

0.99

1.00

1.00

1.00

1.00

0.29

0.19

0.02

0.12

0.01

0.01

Upper diagonal figures are D' and lower diagonal figures are r2.

Pairs in LD (D' > 0.8 or r2 > 0.8) are underlined.

of the protein. Since the Thr98Ser is closely located to Nlinked glycosylation sites, it is possible that the Thr98Ser

polymorphism may affect glycosylation status. Another

possibility is that the Thr98Ser polymorphism may lead to

altered phosphorylation in the VMAT1 protein, since serine and threonine residues play a central role in phosphorylation (activation/inactivation) of proteins. Indeed,

some serine residues have been shown to undergo phosphorylation in the isoform protein VMAT2 [25]. However,

conclusions remain purely speculative and additional

research on protein structure, cell signaling, and protein

trafficking pathways within VMAT1 are required.

Recently, Lohoff et al [30] reported a significant association between the VMAT1 gene and bipolar I disorder.

They genotyped three non-synonymous SNPs (Thr4Pro,

Thr98Ser, and Thr136Ile) and 4 non-coding SNPs, and

found that allele frequencies in the Thr136Ile, and polymorphisms in the promoter region and intron 8 differed

significantly between patients and controls of European

descent. Although the associated SNP was again different

with our results, the results of Lohoff et al [30] and ours

might support the view that schizophrenia and bipolar

has several similarities and share susceptibility genes [31].

Conclusion

We detected a significant association between the VMAT1

gene and schizophrenia only in females. This observation

is not surprising, because there is substantial evidence for

sex differences in the pathogenesis and pathophysiology

of schizophrenia, which may have arisen from interplay

between sex hormones and other developmental factors

[26]. Indeed, there are several other genes (e.g., ZDHHC8

[27] and chimerin 2 [28]) that have been suggested to

have a sexually dimorphic effect on the development of

schizophrenia. Furthermore, there is evidence for crucial

regulation by ovarian steroids on the expression of the

VMAT2 gene [29]. Although there is little information on

such regulation for the VMAT1 gene, it is possible that

similar regulation exists, which may be related to our

observation of the differential effect of the VMAT1 gene

between males and females.

In conclusion, although we failed to replicate the finding

of Bly [22], our results suggest that another amino acid

substitution (Thr98Ser) of the VMAT1 gene may have a

sexually dimorphic effect of giving susceptibility to schizophrenia in the Japanese population. If our results are replicated, further investigations on VMAT1 function may

elucidate molecular mechanisms of schizophrenia, permitting the development of novel therapeutic agents.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

MR, YI, HitK, and TS performed genotyping and statistical

analyses. MR helped to draft the manuscript. HH, KA, and

Table 4: Estimated haplotype frequencies and significance of differences between patients and controls in females

SNP1

C

C

/

C

C

/

C

C

Haplotype

SNP2

SNP3

G

C

C

G

C

C

G

C

/

/

C

C

C

C

C

C

SNP4

/

/

/

/

/

C

C

C

Haplotype frequency (%)

Patients

Controls

0.21

0.13

0.20

0.21

0.14

0.20

0.21

0.14

0.14

0.23

0.29

0.14

0.23

0.29

0.14

0.23

Individual

0.015

0.002

0.003

0.017

0.002

0.004

0.025

0.003

P-values

Global

Permutation global

0.004

0.008

0.012

0.010

0.007

0.011

0.021

0.012

0.012

0.021

Haplotype individual p-values of < 0.05 are listed.

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